The present invention relates generally to methods of treating protozoan infections by administration of bactericidal/permeability-increasing (BPI) protein products.
Protozoa are unicellular eukaryotic organisms that can infect and multiply in mammalian hosts. They may utilize more than one type of host, including insect hosts, during their life cycle. Parasitic protozoa account for a significant portion of all infectious diseases worldwide. Although the majority of protozoan infections occur in developing countries, these infections are seen increasingly in industrialized countries among immigrants and immunosuppressed or immunodeficient individuals. Frequently seen parasitic diseases include malaria, caused by the Plasmodia species, toxoplasmosis, caused by Toxoplasma gondii, leishmaniasis, caused by Leishmania species, and Chagas' disease (American trypanosomiasis), caused by Trypanosoma cruzi. AIDS patients are particularly susceptible to opportunistic protozoan infections such as Pneumocystis carinii and T. gondii. Treatment of protozoan infections is problematic due to lack of effective chemotherapeutic agents, in some instances, or in other instances because of excessive toxicity of the agents and increasingly widespread resistance to the agents.
Malaria is a major health problem in the tropics, and is caused by four Plasmodium species: P. falciparum, P. vivax, P. ovale and P. malariae. The developmental cycle of malaria parasites occurs in female mosquitos, which are the vector for spreading disease, and in humans. Female mosquitos inoculate sporozoites into humans during a blood meal. These sporozoites rapidly enter liver cells, where they develop immediately or after some delay into thousands of individual merozoites. In the relapsing malarias, such as P. vivax and P. ovate, the sporozoites can remain dormant for months to years before entering this proliferative stage. The merozoites rupture from the liver cells and enter the blood stream to invade erythrocytes. These merozoites can either proliferate asexually, or they can differentiate into sexual parasites which then are ingested by the mosquito, where they develop into the infectious sporozoites. After asexual proliferation within the erythrocytes, the merozoites develop through trophozoite forms into the dividing form, the schizont. Each mature schizont contains multiple merozoites which, upon rupture of the infected erythrocyte, are released to invade other erythrocytes and thus continue the cycle.
Clinically, the malaria parasite causes characteristic intermittent fevers and chills, anemia, kidney disease, and brain disease. High levels of parasites in the bloodstream, seen especially in the P. falciparum infection, causes serious complications including severe hemolytic anemia, renal failure, and coma. Thus, diagnosis and early treatment of P. falciparum is crucial. The drug regimen used for treatment of P. falciparum depends on the geographic origin of the infection and the known patterns of drug resistance. Chloroquine resistance is widespread, partial resistance to quinine is seen in many areas, and resistance to the combination of pyrimethamine and sulfadoxine is reported in some areas. Mefloquine is a new anti-malarial that may be effective against chloroquine-resistant P. falciparum. Since treatment failure may occur with any drug regimen, the course of the parasitemia must be followed closely. The non-falciparum parasites are usually treated with chloroquine or amodiaquine, followed by treatment with primaquine if infection is caused by P. vivax or P. ovale.
Toxoplasmosis is a common disease among birds and small mammals, especially cats, that is caused by the protozoan Toxoplasma gondii. About 20 to 70% of adults in this country have positive serologic tests for Toxoplasma infection, depending on the specific population studied. Human infection usually occurs after exposure to infected cat feces or after consuming undercooked meat. Three forms exist in the life cycle of T. gondii: the cyst, the trophozoite and the occyst. The trophozoite is an intracellular form that proliferates during acute infection. Cysts are forms containing several thousand trophozoites that develop within the host cells. They can be seen in any tissue, but are most commonly found in brain and muscle. Oocysts are forms that exist uniquely in the intestines of cats and are shed by cats. After ingestion, the Toxoplasma are liberated from the cysts or oocysts in the gastrointestinal tract. The trophozoites then disseminate through the blood stream or lymphatic system to infect any nucleated host cell.
The vast majority of people who are infected with Toxoplasma have no apparent clinical symptoms, but a small number develop symptoms including enlarged lymph nodes, fever and fatigue. Congenital infection with Toxoplasma has been estimated to cause 20 to 35% of the cases of retinochoroiditis in children and adults. In the immunodeficient patient, toxoplasmosis can appear as a severe disseminated disease that is rapidly fatal. A combination of pyrimethamine and sulfadiazine has been shown to be effective in inhibiting the replication of trophozoites. However, there are no drugs that will kill trophozoites or eradicate the cyst form. Pregnant women cannot be given pyrimethamine because of its potential to cause birth defects. For patients who cannot tolerate sulfadiazine and pyrimethamine, there are no clearly effective alternatives, although some studies suggest that trimethoprim alone or in combination with sulfa drugs may have some anti-toxoplasma activity.
Leishmaniasis is a protozoan infection caused by the genus Leishmania. This parasite exists in two forms: a mobile flagellated form called a promastigote, and a smaller non-mobile, non-flagellated intracellular form, the amastigote. The promastigotes are found in the gut of the sandfly, which is the vector for spreading disease, while amastigotes infect humans and other vertebrate hosts. In the infected animal, Leishmania are found only in macrophages, where they multiply, rupture the host cell, and infect new cells. When Leishmania are ingested by macrophages, they are enclosed within a phagocytic vacuole, where they proliferate. After the macrophages rupture, the amastigotes are taken up by adjacent cells or are transported to distant sites through the bloodstream or lymphatic system.
Visceral leishmaniasis, or kala-azar disease, is caused by the species L. donovani. Primarily affected organs are the liver, spleen, bone marrow and other elements of the reticuloendothelial system, which are enlarged due to the infected macrophages. Other symptoms include fever, weight loss, anemia, and skin lesions. After a few months to a year, the patient becomes emaciated and exhausted. Death is generally due to other concurrent infections. There is also a cutaneous form of leishmaniasis that manifests as ulcerating skin lesions. The drug of choice for treatment of leishmaniasis is pentavalent antimony. Second-line drugs for unresponsive or relapsed patients are pentamidine or amphotericin B. Although orally administered drugs such as rifampin, metronidazole and ketoconazole have been considered for treatment of cutaneous leishmaniasis on the basis of small uncontrolled trials, these drugs are inferior to antimony. Allopurinol analogs are being investigated, but their ultimate usefulness remains to be established.
L. donovani has been found to possess heparin receptors on its cell surface. [Mukhopadhyay et al., Biochem. Journal, 264:517-525 (1989).] These heparin receptors have been localized to the flagellum. [Butcher et al., Experimental Parasitology, 71:49-59 (1990).] It has been shown that protnastigotes and amastigotes of L. donovani bind heparin. Promastigotes in their infective stages bind substantially more heparin than their noninfective counterparts. [Butcher et al., J. Immunol., 148:2879-2886 (1992).] It has also been found that promastigotes in culture produce a cell-associated macromolecule that has characteristics of heparin, indicating that the organism may synthesize heparin or a heparin-like substance. The function of the heparin on the surface of Leishmania may be to facilitate the attachment of parasites to the host phagocytes by serving as a bridge linking the parasite to its host cells, which contain numerous heparin receptors. It has been shown that precoating either parasites or macrophages with heparin augments the interaction of the two cells. Leishmania parasites complete their life cycle within macrophages, which have heparin receptors, but the parasites are killed by neutrophils, which do not bind heparin. Accordingly, the presence of heparin receptors may enhance the likelihood that the parasite will be taken up by a macrophage host cell rather than a neutrophil.
Chagas' disease is an infection caused by Trypanosoma cruzi. It is an incurable multisystemic disease that affects millions of people in Latin America. The prevalence of trypanosomiasis may be as high as 20% of the population in the rural zones of the countries where it is endemic. The parasite exists in three developmental forms: epimastigotes, which multiply extracellularly in the mid-gut of reduviid bugs, the insect vectors for spreading disease; amastigotes, which multiply within mammalian cells; and trypomastigotes, which do not multiply, but transmit infection from the insects to mammals and vice versa. When the insects (reduviid bugs) feed on human blood, the trypomastigotes in their feces penetrate mucous membranes or skin abrasions. The trypomastigotes travel through the blood stream to reach the heart, the gastrointestinal tract and the nervous system, where they invade target cells. After invading the host cell, the trypomastigote transforms into an amastigote, which divides by binary fission. After a number of divisions, amastigotes transform back into trypomastigotes which exit the now ruptured cells, then migrate into neighboring cells or through the blood stream into distant cells. After a period of weeks, the host's immune response suppresses the parasites to very low levels. Small numbers continue to circulate for years.
The acute phase of the disease is characterized by prolonged fever. asthenia, enlarged lymph nodes, edema and hepatosplenomegaly. Afterwards, individuals can remain for many years in a latent or chronic phase. Cardiomyopathy, which occurs in up to 30% of patients, is the most important complication during the chronic phase. Cardiomyopathy is variable in its course; in the acute phase, 10% of the patients die. In the chronic phase, heart disease and heart failure begins in the second to fifth decade and may lead to death. There are two drugs that appear to kill circulating trypanosomes: nifurtimox, a nitrofuran derivative, and benznidazole, a nitroimidazole. T. cruzi strains demonstrate different susceptibilities to these drugs, and both drugs have serious side effects.
Thus, because currently available therapies for protozoan infection are not always effective and may have severe side effects, there remains a need in the art for more effective treatment of protozoan infections.
T. cruzi has been shown to express a unique 60 kD protein on its surface. [Ortega-Barria et al. Cell, 67:411-421 (1991)]. This protein, called penetrin, binds specifically to heparin, heparan sulfate, collagen, and cultured fibroblasts, which are potential host cells of T. cruzi. The ability of T. cruzi trypomastigotes to invade cultured fibroblasts has been shown to be inhibited by penetrin as well as by the glycosaminoglycans heparin and heparan sulfate. The function of penetrin may be to assist the parasites in migrating through the extracellular matrix and to penetrate host cells. Heparin-like sequences and heparan sulfate chains are present on the surface of many potential host cells, including fibroblasts, epithelial cells, glial cells, muscle cells and endothelial cells.
BPI is a protein isolated from the granules of mammalian polymorphonuclear neutrophils (PMNs), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.) A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). BPI is commonly thought to be non-toxic for other microorganisms, including yeast, and for eukaryotic cells. Elsbach and Weiss (1992), supra, reported that BPI exhibits anti-bacterial activity towards a broad range of gram-negative bacteria at concentrations as low as 10.sup.-8 to 10.sup.-9 M, but that 100- to 1,000-fold higher concentrations of BPI were non-toxic to all of the gram-positive bacterial species, yeasts, and eukaryotic cells tested at that time. It was also reported that BPI at a concentration of 10.sup.-6 M or 160 .mu.g/ml had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the gram-positive organisms Staphylococcus aureus (four strains), Staphylococcus epidennidis, Streptococcus faecalis, Bacius subtilis, Micrococcus lysodeikticus, and Listeria monocytogenes. BPI at 10.sup.-6 M reportedly had no toxic effect on the fungi Candida albicans and Candida parapsilosis at Ph 7.0 or 5.5, and was non-toxic to higher eukaryotic cells such as human, rabbit and sheep red blood cells and several human tumor cell lines. See also Elsbach and Weiss, Advances in Inflammation Research, ed. G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms.
The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, the most toxic and most biologically active component of LPS.
In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media [Mannion et al., J. Clin. Invest., 85:853-860 (1990)]. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably result from binding to the anionic groups in the KDO region of LPS, which normally stabilize the outer membrane through binding of Mg.sup.++ and Ca.sup.++. Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabilizaton of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long O-chain bearing, "smooth" organisms being more resistant to BPI bactericidal effects than short O-chain bearing, "rough" organisms Weiss et al., J. Clin. Invest. 65: 619-628 (1980)]. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring the presence of divalent cations and synthesis of new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al., Infection and Immunity 56: 1203-1208 (1988)) but the exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.
There continues to exist a need in the art for new anti-protozoan methods and materials. Products and methods responsive to this need would ideally involve substantially non-toxic compounds available in large quantities by means of synthetic or recombinant methods. Ideal compounds would have anti-protozoan activity when administered or applied as the sole anti-protozoan agent and would also be useful in combinative therapies with other agents.